The present invention relates generally to protein-protein interactions and more specifically to Glutamate Transporter Associated Proteins involved in mediating glutamate transport, chloride transport and cytoskeletal stability and their association with glutamate transporter proteins.
Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system, acting on postsynaptic ionotropic glutamate receptors (particularly NMDA and AMPA receptors). In addition, glutamate stimulates a subset of metabotropic glutamate receptors (particularly the group I metabotropic glutamate receptors mGluR1a and mGluR5) concentrated in the postsynaptic membrane. The timely removal of glutamate from the synaptic cleft is critical to preventing desensitization resulting from continued exposure of the postsynaptic receptors to glutamate. Removal of glutamate from the synaptic cleft is mediated by a class of molecules known as glutamate transporter proteins located on surrounding astroglia and neurons. Five distinct, high-affinity, sodium-dependent glutamate transporters have been identified in animal and human central nervous system. Rat GLAST, GLT-1, EAAC1 (EAAT1, EAAT2 and EATT3, respectively, in human), EAAT4 and EAAT5 differ in structure, pharmacological properties and tissue distribution.
Glutamate transport is a sodium- and potassium-coupled process capable of concentrating intracellular glutamate up to 10,000-fold compared with the extracellular environment. The stoichiometry of the process has been studied and at several models exist proposing various ionic exchanges. In one model derived from salamander retinal glial cells, the transport process is coupled to the co-transport of two sodium ions and the counter-transport of one potassium ion and one hydroxyl ion. (Bouvier et al. (1992), Nature 360:471-474). Another model proposes that with EAAC1, one glutamate is co-transported with three sodium ions and one hydrogen ion, with the counter-transport of one potassium ion (Zerangue et al., Nature (1996) 383:634-637). Yet another study suggests that two sodium ions are co-transported with one glutamate molecule (Hart et al., Science (1998) 280:2112-2114).
The cloning of glutamate transporter subtypes and detailed electrophysiological studies of these proteins reveals that glutamate transporters also possess channel-like properties. The conduct chloride flux is not thermodynamically coupled to substrate transport, although at transportable substrate is required for the chloride conductance. The binding of glutamate to the transporter may change its conformational state to form the chloride channel.
In addition to their possible role in development and learning (due to their potential for modulating normal synaptic transmission), the regulation of synaptic glutamate transporters is likely to play an important role in acute and chronic neurological processes. They can be involved through the disruption of synaptic transmission as well as through glutamate mediated excitotoxicity. Several diseases are associated with disruptions in glutamate transport.
Loss of cerebellar Purkinje cell is the hall mark of several inherited neurodegenerative diseases, including the trinucleotide repeat diseases such as spinocerebellar ataxia type 1 (SCA1), and is commonly associated with neurotoxicity of chronic ethanol ingestion, and with certain paraneoplastic neurological disorders. Although the molecular event that initiates the disease is knownxe2x80x94a trinucleotide repeatxe2x80x94the cellular mechanisms responsible for Purkinje cell degeneration is not known. The selective loss of glutamate transporters such as EAAT4 could make the protein an attractive candidate for a downstream event.
Similarly, dysregulation of glutamate transporter EAAC1 could also have pathological consequences. EAAC1 has the unusual localization to GABA pre-synaptic terminals. This transport could serve as a precursor transporter, supplying extracellular glutamate for GABA re-synthesis. GABA normally is synthesized, via glutamate amino decarboxylase, from glutamate. The source of this glutamate has been traditionally thought to be cellular glutamate. However, the unique localization of the glutamate transporter to GABA terminals suggests that these transporters supply precurser glutamate for GABA re-synthesis. Thus, EAAC1 could serve as an important step in GABAergic neurotransmission. Modulation of GABAergic metabolism is associated with a number of neurological disorders, including epilepsy, tremors, and spasticity. In addition, some theories of schizophrenia include disturbances of glutamate and GABA metabolism.
Accordingly, there is a need in the art for compounds that regulate glutamate transport and in particular, compounds and molecules that interact with glutamate transporter proteins.
The present invention provides a family of proteins that interact with glutamate transporter proteins. Through their interaction with glutamate transporter proteins, Glutamate Transporter Associated Proteins modulate glutamate transport, and also effect cytoskeletal organization and stability as well as chloride flux.
In one embodiment of the invention, there is provided a substantially pure polypeptide characterized as modulating intracellular glutamate transport, interacting with a glutamate transporter protein, and having an expression pattern in the brain. In addition, the polypeptide can have at least one PDZ domain, at least one regulatory G-protein domain, at lest one pleckstrin homology domain, at least one proline-rich domain and at least one guanine exchange factor domain. The polypeptide can have at least one pleckstrin homology domain, at least one spectrin repeat and at least one xcex1-actinin domain.
In an additional embodiment of the invention, there is provided a substantially pure polypeptide characterized as modulating intracellular glutamate transport; interacting with a glutamate transporter protein; having an expression pattern in neural non-neuronal tissues; having at least one kinase C domains; having four transmembrane domains; and being hydrophobic.
In another embodiment of the invention, there is provided a substantially pure polypeptide having an amino acid sequence as set forth in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or conservative variants thereof.
In still another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:2; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:1; (e) degenerate variants of (a), (b), (c) or (d); and (f) a fragment of (a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizes to a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:2.
In yet another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:3; (e) degenerate variants of (a), (b), (c) or (d); and (e) a fragment of (a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizes to a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:4.
In still another embodiment of the invention, there is provided an isolated polynucleotide selected from the group consisting of: (a) a polynucleotide encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:6; (b) a polynucleotide of (a), wherein T can be U; (c) a polynucleotide complementary to (a) or (b); (d) a polynucleotide having a nucleotide sequence as set forth in SEQ ID NO:5; (e) degenerate variants of (a), (b), (c) or (d); and (f) a fragment of (a), (b), (c), (d) or (e) having at least 15 base pairs and hybridizes to a polynucleotide encoding a polypeptide as set forth in SEQ ID NO:6.
In still a further embodiment of the invention, there is provided an antibody that binds to a Glutamate Transporter Associated Protein or binds to immunoreactive fragments thereof. The antibody can be polyclonal or monoclonal.
In yet another embodiment of the invention, there is provided an expression vector comprising a polynucleotide encoding Glutamate Transporter Associated Protein, e.g., SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, or complementary nucleotides thereof and fragments thereof. The vectors can be virus derived or plasmid derived.
In another embodiment of the invention, there is provided a method for producing a Glutamate Transporter Associated Protein polypeptide by culturing a host cell containing a nucleotide encoding a Glutamate Transporter Associated Protein under conditions suitable for the expression of the polypeptide and recovering the polypeptide from the host cell culture.
In another embodiment of the invention, there is provided a substantially pure polypeptide that interacts with the amino acid sequence QEAELTLP (SEQ ID NO:9) or amino acid sequence GRGGNESVM (SEQ ID NO:10).
In still another embodiment of the invention, there is provided a substantially pure polypeptide that interacts with the amino acid sequence set forth in SEQ ID NO:12.
In still another embodiment of the invention, there is provided a substantially pure polypeptide that interacts with the amino acid sequence set forth in SEQ ID NO:13.
In an addition embodiment of the invention, there is provided a method for identifying a compound that modulates a cellular response mediated by a Glutamate Transporter Associated Protein. The method includes incubating the compound with a cell expressing a Glutamate Transporter Associated Protein and a glutamate transporter protein under conditions sufficient to permit the components to interact and comparing a cellular response in the cell incubated with the compound with the cellular response of a cell not incubated with the compound.
In yet another embodiment of the invention, there is provided a method for identifying a compound that inhibits an interaction between a Glutamate Transporter Associated Protein and a glutamate transporter protein. The method includes contacting a Glutamate Transporter Associated Protein with a glutamate transporter protein in the presence of the compound and comparing the formation of a Glutamate Transporter Associated Protein-glutamate transporter protein complex in the presence of the compound with a formation of the complex in the absence of the compound.
In still another embodiment of the invention, there is provided a transgenic non-human animal having a transgene that expresses a Glutamate Transporter Associated Protein chromosomally integrated into the germ cells of the animal. An embodiment of the invention provides a method for producing such transgenic animals.
In another embodiment of the invention, there is provided a computer readable medium having stored thereon a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and sequences substantially identical thereto.
In another embodiment of the invention, there is provided a computer system comprising a processor and a data storage device wherein said data storage device has stored thereon a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and sequences substantially identical thereto.
In yet another embodiment of the invention, there is provided a method for comparing a first sequence to a reference sequence wherein said first sequence is a nucleic acid sequence selected from the group consisting SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 and sequences substantially identical thereto, or a polypeptide sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and sequences substantially identical thereto. The method comprises reading the first sequence and the reference sequence through use of a computer program which compares sequences, and determining differences between the first sequence and the reference sequence with the computer program.
In yet another embodiment of the invention there is provided a method for identifying a feature in a sequence wherein the sequence is selected from the group consisting of a nucleic acid sequence SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 sequences substantially identical thereto, or a polypeptide sequence SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 and sequences substantially identical thereto. The method includes reading the sequence through the use of a computer program which identifies features in sequences and identifying features in the sequences with the computer program.